Fluorescent dyes are known to be particularly suitable for biological applications in which a highly sensitive detection reagent is desirable. Dyes that are able to preferentially bind to a specific biological ingredient or component in a sample enable the researcher to determine the presence, quantity or location of that specific ingredient or component. In addition, specific biological systems can be monitored with respect to their spatial and temporal distribution in diverse environments.
A wide variety of symmetric and asymmetric cyanine dyes and methods for their synthesis have been described, particularly for use in the photographic industry (For example, Brooker et al., J. AM. CHEM. SOC. 73, 5332 (1951); Brooker et al., J. AM. CHEM. SOC. 64, 199 (1942)). Most cyanines possess high visible absorbance and reasonable resistance to photodegradation. Their methods of synthesis permit fine adjustments in their color by changing the aromatic components used for their synthesis or the number of methine groups between the aromatic moieties.
The general structure of cyanine dyes is given by the formula below: ##STR1##
In the above formula X and Y are typically heteroatoms O, S, Se, Te, NR.sup.3, or disubstituted carbon atoms CR.sup.3 R.sup.4, where the substituents on the nitrogen or carbon are typically alkyl groups. Those dyes wherein n=0 are typically referred to as "cyanine" dyes. Where n=1 the dyes are termed "carbocyanine" dyes, while where n=2 the dyes are "dicarbocyanine" dyes and if n=3 the dyes are "tricarbocyanine" dyes, and so on. This class of dyes is generically referred to as "cyanine" dyes no matter the specific number of methine groups between the ring systems. Although the conjugating bridge atoms separating the aromatic rings are typically methine (--CH.dbd.) groups, bridging groups containing rings are known in the art, as are various substituents on the central carbon atom in the spacer. The substituents R.sup.1 and R.sup.2 are typically saturated or unsaturated alkyl groups that are optionally further substituted by a wide variety of other functional groups. The remaining substituents of the cyanine dyes have been shown to include most organic functional groups, as well as additional rings that are fused or not fused rings and that may be additionally substituted themselves.
Those cyanine dyes wherein X.dbd.Y.dbd.O and n=1 are typically known as "oxacarbocyanines", whereas those cyanine dyes wherein X.dbd.Y.dbd.S and n=1 are usually called "thiacarbocyanines" and those dyes wherein X.dbd.Y.dbd.CR.sup.3 R.sup.4 and n=1 are usually called "indocarbocyanines". These classes of cyanine dyes are the most well known, along with those analogs wherein n=0, 2, or 3. For those dyes wherein R.sup.1 and R.sup.2 are simple alkyl chains and all other substituents are H, there exists a widely used naming system. For example, the dye known as DiOC.sub.18 (3) (or "DiO") has X and Y are O, n=1, R.sup.1 and R.sup.2 are octadecyl, and (3) is the total number of methine atoms in the spacer. Similarly, for the analogous dye wherein X and Y are both C(CH.sub.3).sub.2, n=1 and R.sup.1 and R.sup.2 are octadecyl, the common name is DiIC.sub.18 (3) (or "DiI"). The analogous dyes for which X or Y is NR.sup.3, Se and Te are much less common, partially due to a greater degree of synthetic difficulty in their preparation. The counterion .PSI. is typically an anion that balances the intrinsic positive charge of the cyanine dye; however, if the cyanine dye also contains negatively charged groups, the counterion .PSI. may be a cation or the molecule may be an internal zwitterion. In either case .PSI. is present in such a number and with such a total charge as to make the overall molecule electrically neutral.
For most carbocyanines initially described in the literature, the substituents R.sup.1 and R.sup.2 were methyl or ethyl, usually ethyl. However, the use of the lipophilic dyes DiOC.sub.18 (3) and DiIC.sub.18 (3) for staining cellular and synthetic membranes was described as early as 1969 (Czikkely, et al. Z. NATURFORSCH. 24, 1821 (1969)). Since that time the fluorescence properties of these and other structurally related dyes have been used extensively to measure the lateral mobility of the dyes in membranes (Schlessinger et al., SCIENCE 195, 307 (1977)) and more recently to trace neurons in long term cell cultures (Honig et al. J. CELL BIOL. 103, 171 (1986)). In living biological cells, the cyanine dyes in which R.sup.1 and R.sup.2 are both lower alkyl (usually with 6 or less carbon atoms) typically localize in intracellular organelles of live cells such as the mitochondria and the endoplasmic reticulum, where they may be toxic to the cell, whereas the dyes in which R.sup.1 and R.sup.2 are both greater than about 10 carbon atoms typically localize in the cellular membranes, where they are relatively nontoxic to cells. Dyes of this type have been described as being well retained by live cells and liposomes and usually not transferring to adjacent cells or to unlabeled liposomes.
The use of certain cationic lipophilic cyanine dyes, including DiIC.sub.18 (3), DiOC.sub.18 (3) and their C.sub.12 to C.sub.22 homologs in combination with an osmolarity regulating agent to stain cells for the purposes of labeling viable cells, tracking stained cells in vivo, and measuring cell growth rate has been previously described (U.S. Pat. No. 4,762,701 to Horan et al. (1988); U.S. Pat. No. 4,783,401 to Horan et al., (1988); U.S. Pat. No. 4,859,584 to Horan et al. (1989)). However, the labeling procedure used by Horan requires the use of an aqueous "osmolarity-regulating agent" that typically includes sugars, sugar-alcohols, amino acids and "Good's Buffers" in order to keep the dye in solution, as these dyes are generally very insoluble in aqueous solution. Some cell lines, however, may be sensitive to certain osmolarity-regulating agents, particularly sugar-alcohols, and it was therefore advised that users of these dyes conduct standard tests to ensure that cells are viable in the desired osmolarity-regulating agents. Additionally, the presence of inorganic salts, including the ones commonly found in standard buffers such as phosphate-buffered saline (PBS) and in normal culture medium, greatly reduces the solubility of the dyes, thereby lowering the labeling efficiency even in the presence of an osmolarity-regulating agents. Furthermore, many cells do not survive in essentially salt-free media. Finally, like other lipophilic cyanine dyes, the Horan et al. dyes can not tolerate a combined treatment of cell fixation and permeabilization with organic solvents. In histochemical studies, it is common that cells or tissues are fixed with formaldehyde or glutaraldehyde, followed by permeabilization with acetone or alcohol, which usually removes most of the lipids associated with the cell membranes, so that the inside of the cells becomes accessible to macromolecules such as antibodies or labeled antibodies.
Waggoner et al. describes reactive cyanine dyes useful for forming covalent bonds with proteins and other materials (U.S. Pat. No. 5,268,486 to Waggoner et al., (1993)). These particular cyanine dyes are required to contain reactive groups and are required to be sulfonated. However, the Waggoner et al. dyes do not contain lipophilic residues at R.sup.1 and R.sup.2, and are not useful for labeling or studying membranes.